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The objective of this study was to investigate the effects of hydrogen peroxide (H 2 O 2 ) on the chromatin structure of sperm. For this purpose, 44 cryopreserved bovine ejaculates were analyzed immediately post-thaw (control sperm, CON S), after 1 h of post-thaw incubation (non-oxidized sperm, NOX S), and after 1 h of post-thaw incubation in different concentrations of H 2 O 2 (OX S; 50, 100 µM, 1000 µM H 2 O 2 ). Sperm motility was determined using computer-assisted sperm analysis. Sperm plasma membrane and acrosome integrity were assessed by flow cytometry after staining with propidium iodide and fluorescein isothiocyanate-conjugated peanut agglutinin. Chromatin damage was assessed using the sperm chromatin structure assay (SCSA), and DNA damage was evaluated using the single cell gel electrophoresis (Comet) assay. Sperm motility and plasma membrane and acrosome integrity decreased while chromatin damage and DNA damage increased after 1 h of incubation (P < 0.05). The addition of H 2 O 2 adversely affected all parameters (P < 0.05) except for chromatin structure. The role of H 2 O 2 in sperm chromatin damage was investigated by supplementation of 10 IU catalase, which reversed the damage (P < 0.05). Interestingly, the chromatin decondensation induced by 10 mM dithioreitol, evidenced by an increase in chromatin damage in the SCSA (P < 0.05), was reversed by H 2 O 2 . In conclusion, H 2 O 2 causes alterations in sperm functional status and DNA integrity. However, our results show that high concentrations of H 2 O 2 can induce chromatin condensation, and thus the use of the SCSA for the assessment of H 2 O 2 -induced chromatin damage should be carefully considered., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)